Analyzing protein intermediate interactions in living E. coli cells using site-specific photo-crosslinking combined with chemical crosslinking

Information on protein-protein interactions is crucial in understanding protein-mediated cellular processes; however, analyzing transient and unstable interactions in living cells is challenging. Here, we present a protocol capturing the interaction between an assembly intermediate form of a bacterial outer membrane protein and β-barrel assembly machinery complex components. We describe steps for expression of a protein target, chemical crosslinking combined with in vivo photo-crosslinking and crosslinking detection procedures including immunoblotting. This protocol can be adapted to analyze interprotein interactions in other processes. For complete details on the use and execution of this protocol, please refer to Miyazaki et al. (2021).(1)